Microscopic lipid delivery vehicles such as liposomes may be employed to convey therapeutic and diagnostic agents to specific locations in a body. Such delivery vehicles encapsulate the active ingredient, which is thus isolated from degradative effect of body fluids and capable of having a desired effect at particular sites in the body. For this reason, it has long been thought that these vehicles would be appropriate for the therapeutic delivery of oligonucleotides to cells in vivo, since oligos such as DNA and RNA are quickly degraded in body fluids before the therapeutic target cell is reached.
Liposomes are vesicles composed of lipid bilayers completely surrounding an internal aqueous space. They are usually made up of phospholipids or other amphipathic molecules either in pure form or in combination with other molecules such as sterols, i.e., cholesterol. Methods for the preparation and use of liposomes are well known in the art. Such vehicles have been viewed as a potential mechanism for delivering agents to cells to enter, and affect, natural cell growth and other metabolic functions.
In cell metabolism, substances which are normally taken up by cells from the intercellular medium (e.g., vitamins, carbohydrates, amino and nucleic acids, and certain other molecules) cross the cell membrane through natural transporter proteins (i.e., ports) which are specific for the particular substance transported. If a substance does enter the cell through such a natural port, it enters the metabolic pathways of the cell intact. However, substances which cross the cell membrane for which a transporter protein does not exist, such as phospholipid delivery vehicles, go directly to an organelle within the cell called a lysosome. The lysosome is a vacuole which comprises a digestive mechanism for degrading nonspecific nutrients or internal cell products into their primary form for use by the cell.
The liposome delivery of water soluble drugs to cells has often relied upon liposome phagocytosis for transport across the cell membrane, which results in liposome delivery to the lysosome. The lipids are degraded releasing the entrapped drug. For a cytoplasmically or nucleus active drug, the drug must diffuse from this intracellular organelle without degradation to the site of therapeutic action. Certain drugs possess the necessary characteristics to achieve this goal, such as methotrexate or 5-fluoro-oratate, which have a weakly acidic group which upon protonation makes the drug hydrophobic, thus allowing it to pass relatively intact across the lysosomal membrane without loss of therapeutic effect. However, many agents are incapable of escaping lysosomal degradation, and the use of phagocytic transport of liposomal drugs is limited to these specific types of molecules. Oligonucleotides, such as DNA and RNA sequences, require cytoplasmic delivery to be effective and since they are degraded by the lysosome following phagocytic transport by lipid particles.
To attenuate this natural process, liposome formulations have been designed to fuse to internal cellular membranes at low pH and deliver DNA to the cytoplasm of cells. There are several requirements in that the liposome must remain attached to the membrane surface after phagocytosis, and the lipids must be composed of a lipid which forms a fusion competent configuration and which is stabilized by a weakly acidic lipid component. These two requirements once again limit the application of this technology for the extra-lysosomal delivery of agents to cells. First, not all liposome formulations remain attached to the membrane following endocytosis. Second, the fusion competent lipid formulation utilizes lipids which are not ideal for pharmaceutical manufacture, for stable shelf life or for serum stability.
Thus, it has been a desideratum to have a lipid delivery vehicle which would provide for the cytoplasmic delivery of an entrapped active agent in the essential absence of lysosomal degradation, and particularly if such formulations possessed the qualities of shelf life and serum stability. As used herein, the cytoplasmic delivery of an entrapped active agent in the essential absence of lysosomal degradation means a delivery wherein an amount of the active agent sufficient to affect cell function escapes lysosomal degradation and enters the cytoplasm.